PROJECT SUMMARY Post-transcriptional gene regulation by RNA modification is an emerging concept because a diverse set of modified nucleotides are found in mRNA sequences. The overall objective of this proposal is to delineate the molecular and cellular mechanisms by which HIV-1 infection and methamphetamine (MA) alter host transcriptional and translational programs to cause dysfunction of immune system. Our central hypothesis is that HIV-1 infection and use of MA alter the dynamics of the viral and host epitranscriptomes to regulate host-pathogen interactions. Recent findings from the Rana lab support this notion by showing that HIV-1 infection has profound effects on the topology and function of methylated RNAs of both viral and human RNAs. m6A abundance in HIV-1 RNA is controlled by the host methyltransferases METTL3 and METTL14 and the demethylases ALKBH5 and FTO. However, fundamental and critical questions regarding epitranscriptome regulation and its effects on HIV-1 pathogenesis remain to be addressed. To carry out these studies, we developed in vitro infection models as well as biochemical and functional characterization of RNA modifying complexes. Our project has three specific aims: Aim 1: Define the dynamics and mapping of viral and host RNA modifications by HIV infection and methamphetamine (MA) exposure. Aim 2: Determine how RNA modifications influence HIV-1 pathogenesis. Aim 3: Determine the dynamics and specific functions of RNA modifying machinery. Successful completion of these aims will significantly enhance our understanding of the epitranscriptomic regulation of gene expression caused by HIV infection. In addition, these results will provide fundamental understanding of the molecular mechanisms that are altered by MA use leading to immune dysfunctions.